U.S. patent application number 12/376182 was filed with the patent office on 2010-05-27 for method for obtaining oligonucleotide.
This patent application is currently assigned to NEC Soft, Ltd.. Invention is credited to Makio Furuichi, Hiroshi Mizuno, Fumiko Nishikawa, Satoshi Nishikawa, Iwao Waga.
Application Number | 20100129870 12/376182 |
Document ID | / |
Family ID | 38997066 |
Filed Date | 2010-05-27 |
United States Patent
Application |
20100129870 |
Kind Code |
A1 |
Nishikawa; Fumiko ; et
al. |
May 27, 2010 |
METHOD FOR OBTAINING OLIGONUCLEOTIDE
Abstract
The present invention is to provide a method for obtaining an
oligonucleotide such as RNA aptamer having high binding capacity to
a target substance with easy-to-use and high purity. The method for
obtaining oligonucleotide according to the present invention is as
follows: A method for obtaining oligonucleotide comprising the
steps of: performing an electrophoresis of a nucleic acid
molecule/target substance complex comprising a nucleic acid
molecule and a target substance; recovering said nucleic acid
molecule/target substance complex; extracting the nucleic acid
molecule from said nucleic acid molecule/target substance complex;
gene amplifying said nucleic acid molecule.
Inventors: |
Nishikawa; Fumiko;
(Tsukuba-shi, JP) ; Nishikawa; Satoshi;
(Tsukuba-shi, JP) ; Furuichi; Makio; (Koto-ku,
JP) ; Mizuno; Hiroshi; (Koto-ku, JP) ; Waga;
Iwao; (Koto-ku, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
NEC Soft, Ltd.
Koto-ku, Tokyo
JP
National Institute of Advanced Industrial Science and
Technology
Chiyoda-ku, Tokyo
JP
|
Family ID: |
38997066 |
Appl. No.: |
12/376182 |
Filed: |
July 10, 2007 |
PCT Filed: |
July 10, 2007 |
PCT NO: |
PCT/JP2007/063741 |
371 Date: |
February 3, 2009 |
Current U.S.
Class: |
435/91.1 ;
204/462 |
Current CPC
Class: |
C12N 2330/30 20130101;
C12N 15/115 20130101; C12N 2310/16 20130101 |
Class at
Publication: |
435/91.1 ;
204/462 |
International
Class: |
C12P 19/34 20060101
C12P019/34; B01D 57/02 20060101 B01D057/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2006 |
JP |
2006-212046 |
Claims
1. A method for obtaining oligonucleotide comprising the steps of:
performing an electrophoresis of a nucleic acid molecule/target
substance complex comprising a nucleic acid molecule and a target
substance; recovering said nucleic acid molecule/target substance
complex; extracting the nucleic acid molecule from said nucleic
acid molecule/target substance complex; gene amplifying said
nucleic acid molecule.
2. The method for obtaining oligonucleotide according to claim 1,
wherein said step of performing the electrophoresis is a step of
performing an electrophoresis at least using an agarose gel.
3. The method for obtaining oligonucleotide according to claim 1,
wherein said method for obtaining oligonucleotide comprises the
step of performing the electrophoresis using the agarose gel and
the step of performing the electrophoresis using a polyacrylamide
gel.
4. The method for obtaining oligonucleotide according to claim 1,
further comprising a step of contacting said nucleic acid molecule
and said target substance, prior to the step of performing the
electrophoresis.
5. The method for obtaining oligonucleotide according to claim 1,
wherein said oligonucleotide is RNA aptamer.
6. The method for obtaining oligonucleotide according to claim 1,
wherein said target substance is protein
Description
FIELD OF INVENTION
[0001] The present invention relates to a method for obtaining
oligonucleotide.
RELATED ART
[0002] Although it has believed that oligonucleotides such as DNA
and RNA mainly have a function as molecules concerning the
synthesis of proteins, it has numerously found that molecules
perform the expression of function thereof by means of the
interaction with gene, e.g. ribozyme and RNAi. Attention is
currently focused on the application for therapeutical field.
Especially, the aptamer has been paid attention as a nucleic acid
which binds to macromolecule such as proteins capable of altering
the function of the macromolecule. In order to apply it to
medicines, new aptamers have been obtained.
[0003] The aptamer such as RNA aptamer has been generally obtained
in accordance with SELEX method (Systematic Evolution of Ligands by
EXponential enrichment) (see Patent Document 1). In case of
obtaining RNA aptamer with SELEX method, a target substance such as
protein is associated with RNA library comprising randomized
sequence to form a complex of RNA/target substance. Next, the
complex of RNA/target substance is attached on a protein-adsorptive
filter such as nitrocellulose filter. Free RNA which is not
involved to form the complex is included on the filter, in addition
to the complex of RNA/target substance. The free RNA is physically
washed from the filter by using a buffer. The filter is treated
with a buffer containing any agent including urea to obtain a
desired RNA from the complex of RNA/target substance which is
included on the filter. The obtained RNA is amplified with PCR
methods to obtain a gene product, and the above-mentioned steps of
the formation of complex, and the washing and treatment through the
filter are repeated. The SELEX method is a method for obtaining
gene products which can specifically bind to target substance by
means of repeating such steps.
[0004] In the method for obtaining the oligonucleotide such as RNA
aptamer in accordance with the SELEX method, it is necessary to
attach a mixture containing the complex of oligonucleotide/target
substance which is obtained to associate an oligonucleotide library
such as RNA library with the target substance, to the
protein-adsorptive filter such as nitrocellulose filter. This will
result in binding the complex of oligonucleotide/target substance
on the filter. In addition, the oligonucleotide which cannot be
bound to the target substance, that is, the oligonucleotide not to
be subjected to obtain with this method will be also attached on
the filter by means of non-specific adsorption it with the filter.
Accordingly, in order to only obtain the desired oligonucleotide
from the complex of oligonucleotide/target substance, it is
necessary to further perform steps of washing the filter so as not
to remain an undesired oligonucleotide on the filter and to remain
the complex of oligonucleotide/target substance on the filter and
of extracting the desired oligonucleotide from the filter.
Therefore, it is difficult to efficiently obtain the desired
oligonucleotide by means of the above-mentioned washing step and
extracting step due not only to the interaction of the undesired
oligonucleotide with the filter but also to the interaction of the
undesired oligonucleotide with the complex of
oligonucleotide/target substance.
[0005] In addition, it is necessary to repeat each step as
mentioned above with several to ten or more times in the SELEX
method in order to obtain the oligonucleotide having high binding
capacity to the target substance. So, it has a disadvantage in view
of cost and operating efficiency.
[0006] Further, it is necessary to use an apparatus including the
filter manifold for the above-mentioned washing/extracting step.
The method is unsuitable for obtaining the oligonucleotide with
large scale.
Patent Document 1
[0007] JP 2763958B
DISCLOSURE OF THE INVENTION
Problems to be Solved
[0008] The present invention is based on such a problem, and is to
provide a method for obtaining an oligonucleotide such as RNA
aptamer having high binding capacity to a target substance with
easy-to-use and high specificity.
Means to Solve the Problems
[0009] The method for obtaining oligonucleotide according to the
present invention is as follows:
[0010] A method for obtaining oligonucleotide comprising the steps
of:
[0011] performing an electrophoresis of a nucleic acid
molecule/target substance complex comprising a nucleic acid
molecule and a target substance;
[0012] recovering said nucleic acid molecule/target substance
complex;
[0013] extracting the nucleic acid molecule from said nucleic acid
molecule/target substance complex;
[0014] gene amplifying said nucleic acid molecule.
EFFECT OF INVENTION
[0015] In accordance with the present invention, it is possible to
obtain the oligonucleotide having high binding capacity with an
advantage in cost and in a short time.
[0016] In addition, it is possible to obtain such an
oligonucleotide in bulk with small scale, which is impossible in
the prior art.
BRIEF EXPLANATION OF DRAWING
[0017] FIG. 1 is an electrophoresis image as obtained in the
Reference Example 1.
[0018] FIG. 2 is an electrophoresis image as obtained in the
Reference Example 2.
[0019] FIG. 3 is a visualized image as obtained in the Embodiment 2
and the Comparative Examples 1 and 2.
[0020] FIG. 4 is an autoradiogram as obtained in the Embodiment 2-1
at second generation.
[0021] FIG. 5 is an autoradiogram as obtained in the Embodiment 2-1
at third generation.
[0022] FIG. 6 is an autoradiogram as obtained in the Embodiment 2-1
at fourth generation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0023] <The Method for Obtaining Oligonucleotide According to
the Present Invention>
[0024] The method for obtaining oligonucleotide according to the
present invention is as follows:
[0025] A method for obtaining oligonucleotide comprising the steps
of:
[0026] performing an electrophoresis of a nucleic acid
molecule/target substance complex comprising a nucleic acid
molecule and a target substance;
[0027] recovering said nucleic acid molecule/target substance
complex;
[0028] extracting the nucleic acid molecule from said nucleic acid
molecule/target substance complex;
[0029] gene amplifying said nucleic acid molecule.
Hereinafter, the "step of performing an electrophoresis of a
nucleic acid molecule/target substance complex comprising a nucleic
acid molecule and a target substance" is referred to as an
electrophoresis step, the "step of recovering said nucleic acid
molecule/target substance complex" is referred to as a recovery
step, the "step of extracting the nucleic acid molecule from said
nucleic acid molecule/target substance complex" is referred to as
an extraction step and the "step of gene amplifying said nucleic
acid molecule" is referred to as an amplification step.
[0030] The method for obtaining oligonucleotide according to the
present invention is a method for obtaining the oligonucleotide
which can be bound to the target substance from the nucleic acid
molecule wherein it performs the steps of the electrophoresis step,
the recovery step, the extraction step and the amplification step
to obtain the gene product such as RNA and DNA, and if necessary,
the obtained gene product is subjected to perform the steps
including the electrophoresis step and the following steps.
[0031] (The Electrophoresis Step)
[0032] The electrophoresis step in the method for obtaining
oligonucleotide according to the present invention is performed
such that the mixture containing nucleic acid molecule such as
single strand RNA and nucleic acid molecule/target substance
complex comprising the target substance such as proteins is
introduced into an appropriate substrate such as agarose gel and a
voltage is applied to the substrate. This will result in separating
the nucleic acid molecule and the nucleic acid molecule/target
substance complex contained in the mixture due to the difference in
mobility in the substrate.
[0033] In the method for obtaining oligonucleotide according to the
present invention, the electrophoresis step is performed with at
least 1 to 3 times, especially, preferably with once in the agarose
gel in view of efficiently obtaining the oligonucleotide. In
addition, in the method for obtaining oligonucleotide according to
the present invention, the agarose gel is preferably used for the
first time of the electrophoresis step. This will enable to
efficiently remove the nucleic acid molecule such as RNA molecule
which non-specifically binds. Further, in the method for obtaining
oligonucleotide according to the present invention, the method
preferably comprises the electrophoresis step using the agarose gel
and the electrophoresis step using the polyacrylamide gel. This
will enable to fractionate and obtain candidates to be subjected
such as aptamer with easy-to-handle and in a short time. Such a
candidate can be specifically fractionated and obtained in the
polyacrylamide gel method.
[0034] [The Nucleic Acid Molecule]
[0035] In the present invention, the nucleic acid molecule
generically refers to as a so-called nucleic acid including
adenine, guanine, cytosine, thymine and uracil, and/or so-called
gene-related substances of nucleoside comprising nucleic acid
analog substance in which the sugar portion of the nucleoside is
ester-linked with the phosphate residue thereof. For example,
example of the nucleic acid molecule includes single strand RNA,
single strand DNA, double strand DNA and double strand RNA.
Hereinafter, it will be described with regard to the single strand
RNA (RNA aptamer) to be subjected to obtain.
[0036] In this case, the nucleic acid molecule may comprise: a
region containing randomized sequence consisting of A, G, C and U
(hereinafter, referred also to as a random region); and a fixed
sequence containing a known sequence at 5'-end and 3'-end of the
random region (hereinafter, referred also to as a 5'-end fixed
sequence and a 3'-end fixed sequence, respectively). These fixed
sequences correspond to a primer region necessary for PCR methods
for performing the amplification step as mentioned below in the
method for obtaining oligonucleotide according to the present
invention.
[0037] Such a nucleic acid molecule can be easily obtained as the
following method. That is, a gene product comprising a
complementary DNA to the above-mentioned single strand RNA (5'-end
fixed sequence-random region-3'-end fixed sequence) may be
preliminary prepared, and the object nucleic acid molecule may be
prepared from such a gene product with DNA-dependent RNA polymerase
such as T7 RNA polymerase in accordance with in vitro transcription
method. In this case, a sequence capable of activating the promoter
activity of the DNA dependent RNA polymerase as used may be linked
to the upstream of the complementary sequence to the 5'-end fixed
sequence.
[0038] The 5'-end fixed sequence and 3'-end fixed sequence of the
nucleic acid molecule is not limited so far as it has less effect
for binding the nucleic acid molecule and target substance and it
provides a primer region necessary for PCR methods in the
amplification step. In case of using single strand RNA as the
nucleic acid molecule, the 3'-end fixed sequence will offer the
primer region for RNA dependent DNA polymerase and DNA dependent
DNA polymerase in the amplification step, and the 5'-end fixed
sequence will offer the primer region for DNA dependent DNA
polymerase in the amplification step. Accordingly, the 5'-end fixed
sequence and 3'-end fixed sequence may be appropriately selected
based on the expression profile of the activity of these
polymerases.
[0039] The random region of the nucleic acid molecule is a gene
comprising A, G, C and U (in the sequence listing, correctively
referred to as "n") having a predetermined base length. The base
length may be appropriately altered based on the oligonucleotide to
be subjected to obtain and on the property of the target substance,
and may be, for example, 15 to 60 bases.
[0040] A method for manufacture of the random region is not
limited, including artificial synthesis, semi-artificial synthesis.
For example, example of such a method includes a synthesis method
using nucleic acid synthesis apparatuses such as 3400 DNA
synthesizer (Applied Biosystems), an extraction method according to
the well known method such as the phenol extraction of natural
origin gene products, and a preparation method of cleaving such a
gene product with gene cleavage enzymes.
[0041] It should be noted that the present invention is not limited
to use the single strand RNA, although the above-mentioned
description is for the single strand RNA as one of examples of the
nucleic acid molecule used in the method for obtaining
oligonucleotide according to the present invention. With regard to
the other molecular species, the nucleic acid molecule can be
prepared by appropriately selecting the sequence, primer and
enzymes and the others based on the molecular species.
[0042] [The Target Substance]
[0043] The target substance means a counterpart substance to be
obtained in the present invention. Example of the target substance
includes molecules, compounds and substances which can form a
complex with nucleic acid molecule by means of the intermolecular
associations such as hydrogen bonding and ion binding with the
oligonucleotide. For example, example of the target substance
includes peptides, proteins, glycoproteins, hormones, growth
factors, receptors, antigens, antibodies, nucleic acids,
polysaccharides, carbohydrates and synthetic compounds.
[0044] [The Nucleic Acid Molecule/Target Substance Complex]
[0045] The nucleic acid molecule/target substance complex
comprising the nucleic acid molecule and the target substance is
prepared by incubating the nucleic acid molecule and the target
substance in buffers such as Tris-based or in an appropriate
solvent for a predetermined period. In this case, for the purpose
of efficiency of the electrophoresis step, high density solution
such as glycerol is preferably added. Example of such an additive
includes glycerol and saccharose solution. The amount of the
additive is not limited so far as the formation of the nucleic acid
molecule/target substance complex is not inhibited. For example,
the amount is preferably in the range of 10 to 30 w/v % (10 to 30
v/v % with regard to the volume of the reaction mixture). In
addition, for the purpose of facilitating the formation of the
nucleic acid molecule/target substance complex, monovalent or
divalent cationic ion may be added.
[0046] A method for preparing the nucleic acid molecule/target
substance complex, but not limited to, may be such that the nucleic
acid molecule and the target substance is added to and in contact
with the above-mentioned appropriate solvent. Such a contact is
performed at which the substrate is introduced, in case of
performing the electrophoresis step. In addition, it may be
performed in an appropriate container.
[0047] [The Substrate]
[0048] In the present invention, the substrate used in the
electrophoresis step is not limited to so far as the nucleic acid
molecule and the nucleic acid molecule/target substance complex
contained in the mixture can be electrically separated. Example of
the substrate includes agarose gel, polyacrylamide gel and
water-soluble cellulose gel. The concentration of the substrate is
not limited to so far as the nucleic acid molecule and the nucleic
acid molecule/target substance complex can be separated in the
substrate. For example, in case of using agarose as the substrate,
the concentration of the substrate may be in the range of 0.5% to
2.0%, preferably 0.7% to 1.5%. In addition, in case of using
polyacrylamide gel as the substrate, the concentration of the
substrate may be in the range of 5 to 20%, preferably 7 to 12%.
[0049] In the electrophoresis step, the substrate may be prepared
to solve in buffers capable of electrically charging the nucleic
acid molecule, the nucleic acid molecule/target substance complex
and the others. A well-known buffer which can be used for the
electrophoresis may be used, including Tris-based and HEPES-based
buffer. Among them, in case of performing the electrophoresis step
using agarose gel, example of the buffer includes TBE buffer
(Tris-borate-EDTA buffer) and TAE buffer (Tris-acetate-EDTA
buffer).
[0050] The electrophoresis in the electrophoresis step may be
performed such that the binding condition of the nucleic acid
molecule/target substance complex is maintained in the substrate
and an appropriate voltage is applied for the mixture introduced in
the substrate so as to be capable of transferring the nucleic acid
molecule and the nucleic acid molecule/target substance complex in
the substrate with different mobility. The range of the voltage and
the applied time may be appropriately selected in view of the
mobility of the mixture in the substrate. For example, in case of
performing the electrophoresis step using the agarose gel, the
electrophoresis can be performed for 5 to 30 minutes at the voltage
of 50 to 150V. In case of performing the electrophoresis step using
the polyacrylamide gel, the electrophoresis can be performed for 1
to 4 hours at the voltage of 100 to 200 V. The applied time may be
that the nucleic acid molecule/target substance complex and the
nucleic acid molecule or the target substance which is not involved
in the formation of the nucleic acid molecule/target substance
complex can be electrophoretically separated. In addition, the
temperature of the electrophoresis step is not limited so far as
the mobile phase is not solidified, and may be in the range of
2.degree. C. to room temperature.
[0051] The mobile phase performing the electrophoresis step may be
used so far as the binding condition of the nucleic acid
molecule/target substance complex is at least maintained, including
a solvent containing a well-known buffer such as Tris. If
necessary, electrolytes such as cationic ion and anionic ion may be
added in the mobile phase. The mobile phase may contain monovalent
or divalent cationic ion in view of maintaining the binding
condition of the nucleic acid molecule/target substance
complex.
[0052] So separated nucleic acid molecule/target substance complex
from the nucleic acid molecule in the substrate is provided in the
following recovery step.
[0053] (The Recovery Step)
[0054] In the method for obtaining oligonucleotide according to the
present invention, the recovery step is a step of recovering the
nucleic acid molecule/target substance complex from the substrate
as obtained in the electrophoresis step. The recovery step may be
appropriately selected in accordance with the existence form of the
nucleic acid molecule/target substance complex in the substrate.
For example, example of means for carrying out the recovery step
includes a method of directly inserting an appropriate dispensing
apparatus or instrument such as pipette into the well of the
substrate to collect the subject, or a method of cutting the
substrate containing nucleic acid molecule/target substance complex
with cutter to recover the subject as a piece of the substrate.
[0055] The nucleic acid molecule/target substance complex contained
in the substrate after the electrophoresis step may be located in
and near the well in which the mixture is introduced in the
substrate, or may form the band shape in the substrate, depending
on the condition of the electrophoresis.
[0056] In case of locating the nucleic acid molecule/target
substance complex in the well, using the appropriate dispensing
means such as pipette, the means may be inserted in the well to
directly collect the nucleic acid molecule/target substance
complex. This technique may be applied in case of locating the
nucleic acid molecule/target substance complex near the well. In
this case, the nucleic acid molecule/target substance complex will
be recovered in the solution state.
[0057] Even in case of locating the nucleic acid molecule/target
substance complex near the well, when the above-mentioned technique
is difficult to use, the substrate near the well may be cut into
the piece of the substrate having an appropriate size to collect
the nucleic acid molecule/target substance complex along with the
substrate. This technique can be applied in case that the nucleic
acid molecule/target substance complex forms the band shape in the
substrate. That is, in case of locating the nucleic acid
molecule/target substance complex in the substrate, a part of the
substrate containing the nucleic acid molecule/target substance
complex may be cut with cutter to recover the nucleic acid
molecule/target substance complex along with the substrate.
[0058] In the recovery step, one of the above-mentioned techniques
or an appropriate combination thereof may be selected.
[0059] In the recovery step, depending on the property of the
nucleic acid molecule and/or target substance to be subjected, an
appropriate method capable of observing these molecules and/or
substances may be appropriately used as a method for confirming the
existence of the nucleic acid molecule/target substance complex in
the substrate.
[0060] For example, example of the method of confirming the
existence of the nucleic acid molecule in the substrate includes a
method of visualizing the molecule using a well-known detecting
agent for the gene product such as ethidium bromide and cybergreen.
In addition, it may be that the nucleic acid molecule is
preliminary labeled with radioactive compounds such as .sup.32P-ATP
or with fluorescence agent such as fluorescein and Cy5, and these
labels are detected.
[0061] In addition, in case of using proteins as the target
substance, example of the method of confirming the existence of the
target substance in the substrate includes a method for visualizing
the protein such as silver staining, coomassie brilliant blue (CBB)
staining.
[0062] So recovered nucleic acid molecule/target substance complex
will be provided in the following extraction step.
[0063] (The Extraction Step)
[0064] In the method for obtaining oligonucleotide according to the
present invention, the extraction step is a step of extracting the
nucleic acid molecule from the nucleic acid molecule/target
substance complex recovered in the recovery step. The extraction
step may be appropriately performed in accordance with the
existence form (e.g. the solution state, and a state along with the
substrate) of the nucleic acid molecule/target substance complex
obtained in the recovery step. Example of the recovery step
includes a well-known method of recovering gene products such as
phenol extraction, ethanol precipitation and spin column
method.
[0065] In case that the nucleic acid molecule/target substance
complex has been recovered in the solution state, the nucleic acid
molecule may be extracted from the nucleic acid molecule/target
substance complex using the phenol extraction and ethanol
precipitation and the others. In case that the nucleic acid
molecule/target substance complex has been recovered along with the
substrate, the nucleic acid molecule may be extracted from the
nucleic acid molecule/target substance complex using the spin
column having an appropriate cut off value based on the molecular
weight and other property of the target substance.
[0066] So extracted nucleic acid molecule will be provided in the
following amplification step.
[0067] (The Amplification Step)
[0068] In the method for obtaining oligonucleotide according to the
present invention, the amplification step is a step of amplifying
the nucleic acid molecule obtained in the extraction step and the
other steps. The amplification step is a step of the gene
amplification using PCR methods such as RT-PCR, PCR and in vitro
transcription. The amplification step may be performed, by
utilizing the fixed sequence contained in the 5'-end and 3'-end of
the nucleic acid molecule, with the primer which complementary
binding to these fixed sequences. As an example, the amplification
step using the single strand RNA will be explained.
[0069] First, the single strand RNA obtained in the extraction
step, RNA dependent DNA polymerase (reverse transcriptase) such as
reverse transcriptase originated from avian myeloblastosis virus
(AMV Reverse Transcriptase), and the primer complementary to the
fixed sequence at 3'-end of the single strand RNA are incubated to
amplify the complementary DNA to this single strand RNA. So
amplified complementary DNA are further amplified into a double
strand DNA by using primers complementary to the 5'-end fixed
sequence and the 3'-end fixed sequence contained in the
complementary DNA and a well-known DNA dependent DNA polymerase
such as Taq DNA polymerase. Then, the double strand DNA is
transcribed into the single strand RNA with a well-known DNA
dependent RNA polymerase such as T7 RNA polymerase.
[0070] In this way, the subject nucleic acid molecule capable of
binding to the target substance are specifically amplified through
the steps of the electrophoresis step, the recovery step, the
extraction step and the amplification step.
[0071] Although the single strand RNA is exemplified as the nucleic
acid molecule, the amplification step may be appropriately
performed by selecting polymerases and primers in accordance with
the subject nucleic acid molecule to be amplified, in case of using
the other nucleic acid such as single strand DNA, double strand RNA
and double strand DNA as the nucleic acid molecule.
[0072] So transcribed nucleic acid molecule may be as the
oligonucleotide obtained in the method for obtaining
oligonucleotide according to the present invention. In addition,
the nucleic acid molecule may be subjected to repeating the
above-mentioned steps, if necessary.
[0073] (The Other Steps)
[0074] The method for obtaining oligonucleotide according to the
present invention may comprise a step of using the filter in
accordance with the above-mentioned SELEX method in addition to the
electrophoresis step. The step of using the filter in accordance
with SELEX method is the same in the prior art.
EMBODIMENT
Reference Example 1
[0075] 200 nM of oligonucleotide (sequence number 1), and 0, 200
nM, 2 .mu.M and 4 .mu.M of .DELTA.NS3 protein (sequence number 2)
originated from NS3 protease of hepatitis virus C wherein the
His-Tag having 6 histidine residues is liked to the N terminal of
the protein (hereinafter, referred simply to as .DELTA.NS3 protein)
were incubated in a binding buffer solution (20 .mu.L of reaction
volume) at 20% final concentration of glycerol (50 mM of Tris-HCl
(pH7.8), 5 mM of MgCl.sub.2 and 5 mM of CaCl.sub.2) for 20 minutes
at room temperature.
[0076] So obtained mixture was introduced in 1% of agarose gel
containing 0.00005% of ethidium bromide, and an electrophoresis was
performed in TBE buffer (44.5 mM Tris, 44.5 mM of boric acid and
EDTA (1 mM, pH 8.0)) for 7 minutes at 100V. The gel was immersed in
1% ethidium bromide solution and was observed under UV illuminator.
The result is shown in FIG. 1. In FIG. 1, lane 1 corresponds to
tRNA, lane 2 corresponds to control, and lanes 3 to 6 correspond to
mixtures containing 200 nM of oligonucleotide and .DELTA.NS3
protein wherein the concentrations of .DELTA.NS3 protein are 0, 200
nM, 2 .mu.M and 4 .mu.M, respectively.
Reference Example 2
[0077] The Reference Example 2 was performed similar to the
Reference Example 1, except that 2 .mu.M of .DELTA.NS3 protein and
0, 200 nM, 1 .mu.M and 2 .mu.M of tRNA (Product name: 10 109 541
001 (Roche)) were used to contain in the mixture, instead of 0, 200
nM, 2 .mu.M and 4 .mu.M of .DELTA.NS3 protein, and the gel was
observed. The result is shown in FIG. 2. In FIG. 2, lane 1
corresponds to tRNA, lane 2 corresponds to control, and lanes 3 to
6 correspond to mixtures containing 200 nM of oligonucleotide, 200
nM of .DELTA.NS3 protein and tRNA wherein the concentrations of
tRNA are 0, 200 nM, 1 .mu.M and 2 .mu.M, respectively.
Embodiment 1
[0078] The following [Gel method] was performed for fifth
generation, using 200 nM of .DELTA.NS3 protein as the target
substance. Then, the double strand DNA products at each generation
were subjected to the following [Radiolabeling] to obtain
radiolabeled-in vitro transcripts. The transcripts and 0 nM (no
proteins), 50 nM and 100 nM of .DELTA.NS3 protein were subjected to
the following [Binding Experiment]. The obtained radioactive
intensities are shown in Table 1.
Embodiment 2
[0079] The following [Gel method] was performed for first
generation, using 1 .mu.M of mouse IgG (Catalog No. 3D3 (Hytest))
as the target substance, then the following [SELEX method] was
performed for third generation. Then, the following [Radiolabeling]
was performed, using the double strand DNA product obtained in the
last generation, to obtain a radiolabeled in vitro transcript. The
[Binding Experiment] was performed, using the transcript and 0 nM,
50 nM and 100 nM of mouse IgG, 0 nM, 50 nM and 100 nM of
maltose-binding protein (MBP) (Catalog No. E8044S (New England
BioLab)) and 0 nM, 50 nM and 100 nM of purified
glutathione-S-transferase (GST) (prepared in our laboratory). The
obtained visualized image of the radioactive intensity is shown in
FIG. 3, and the values thereof are shown in Table 2.
Comparative Example 1
[0080] The following [SELEX method] was performed for fifth
generation, using 1 .mu.M of mouse IgG as the target substance.
Then, the following [Radiolabeling] was performed, using the double
strand DNA product as obtained at the last generation, to obtain a
radiolabeled in vitro transcript. The following [Binding
Experiment] was performed, using the transcript and the target
protein as mentioned in the Embodiment 2. The obtained visualized
image of the radioactive intensity is shown in FIG. 3 and the
values thereof are shown in Table 2.
Comparative Example 2
[0081] The Comparative Example 2 was performed as the same as the
Comparative Example 1, except that the following [SELEX method] was
performed for seventh generation instead of fourth generation in
the Comparative Example 1. The result is shown in FIG. 3 and Table
2.
[0082] [Preparation of RNA Pool]
[0083] The DNA as shown in the sequence number 3 was synthesized
with DNA synthesizer (334 DNA synthesizer (Applied Biosystems)).
This pool (about 10 .mu.M) and a base sequence of a primer
comprising 5'-end of T7 promoter sequence (sequence number 4) were
reacted in the presence of the DNA polymerase to link the T7
promoter. Then, a transcriptional reaction was performed, using T7
RNA polymerase with so obtained double strand DNA as the template
to obtain RNA pool (sequence number 5).
[0084] [Gel Method]
[0085] 200 nm to 20 .mu.M of the RNA pool was added to the binding
buffer solution and stood for 5 minutes at room temperature. Then,
to the solution, a metal ion (Mg2+, final concentration of 5 mM)
and glycerol was added to prepare its concentration as 20%, and the
target substance was added, and stood for 10 minutes at room
temperature.
[0086] The mixture was introduced into wells of 1% agarose gel
which is preliminary cut at the circumference of the well, and the
electrophoresis was performed for 7 minutes at 100 V in TBE buffer
solution.
[0087] After the electrophoresis, the solution in the well was
recovered, and the oligonucleotide solution was recovered, from the
agarose gel piece which is preliminary cut, with spin column
(Product Name: Ultrafree-DA (Millipore)). The solution in the well
and the oligonucleotide solution obtained from the agarose gel
piece were subjected to ethanol precipitation to obtain an
oligonucleotide.
[0088] The steps as mentioned above and the following steps from
the following [RT] to the following [in vitro transcription] are
correctively referred to as a first generation. In addition, each
step of the next generation was performed, using the in vitro
transcript as obtained in the just before generation, instead of
the RNA pool.
[0089] [SELEX Method]
[0090] 20 .mu.M of the RNA pool and the target substance were mixed
in the binding buffer solution, and the mixture was introduced in
the nitrocellulose membrane fixed to the pop top holder to filter
the mixture, and the membrane was washed with 1 mL of the binding
buffer solution. Then, the membrane was immersed in 200 .mu.L of an
eluting solution (0.4 M of sodium acetate, 5 mM of EDTA and 7 M of
urea (pH5.5)), and heated for 5 minutes at 90.degree. C. So
obtained solution was subjected to the ethanol precipitation to
obtain an oligonucleotide.
[0091] The steps as mentioned above and the following steps from
the following [RT], the following [PCR] and the following [in vitro
transcription] in which the oligonucleotide is used are
correctively referred to as a first generation of SELEX method. In
addition, each step of the next generation was performed, using the
in vitro transcript as obtained in the just before generation,
instead of the RNA pool.
[0092] [RT]
[0093] The reverse transcription reaction was performed for 1 hour
at 42.degree. C. with all of the oligonucleotides as recovered,
primer 1 (sequence number 6) and Reverse-iT RTase Blend
(ABgene).
[0094] [PCR]
[0095] The PCR reaction was performed for 18 cycles, using the
whole volume of the reaction product of the reverse transcription
reaction, 60 pg of primer 2 (sequence number 7) and primer 3
(sequence number 8) with gene Taq enzyme (Nippon Gene) to obtain a
double strand DNA product, wherein one cycle of the 18 cycles
corresponds to reactions for 30 seconds at 95.degree. C., for 30
seconds at 55.degree. C. and for 30 seconds at 72.degree. C.
[0096] [In Vitro Transcription]
[0097] The in vitro transcription reaction was performed, using the
double strand DNA product (0.5 .mu.g) as obtained in the PCR
reaction and T7 RNA polymerase (Product Name: Ampliscribe
(EPICENTRE), volume used 1 .mu.L) to obtain an in vitro
transcript.
[0098] [Radiolabeling]
[0099] The above-mentioned [in vitro transcription] was performed,
using the above-mentioned double strand DNA product in the presence
of .alpha.-.sup.32P-ATP (Amersham Biosciences) to obtain a
radiolabeled in vitro transcript.
[0100] [Binding Experiment]
[0101] The radiolabeled in vitro transcript and the target protein
were incubated in the binding buffer solution for 10 minutes at
room temperature.
[0102] The obtained mixture was introduced on the filter (Product
Name: MF-membrane filter (Millipore)) which is sucked with the
sucker, the filter was then washed with 20 times volume of the
binding buffer solution with regard to the mixture. The radioactive
intensity of so obtained filter was measured with the Bioimaging
analyzer (BAS-2500, FUJIFILM (using BAS-MS2040 as the imaging
plate)). The radioactive intensity was visualized with ImageReader
(Id.), and so obtained date was quantified with ImageGauge ver. 4.0
(Id.).
TABLE-US-00001 TABLE 1 .DELTA.NS3 protein Generation 0 nM 50 nM 100
nM Zero 0.1 4.9 8.1 First 1.2 11.5 29.4 Second 1.4 16.1 20.8 Third
1.3 17.7 20.9 Fourth 0.5 9.7 15.4 Fifth 1.8 11.7 26.4
TABLE-US-00002 TABLE 2 Protein 0 nM 50 nM 100 nM Embodiment 2 0.1
2.4 4.6 Comparative Example 1 0.1 0.3 0.4 Comparative Example 2 0.2
1.7 2.4
[0103] The values as shown in Tables 1 and 2 indicate a percentage
in case that the radioactive intensity of 1/5 volume of the
radiolabeled in vitro transcript before subjecting to the [Binding
Experiment] is set as 20%.
[0104] The gene sequence of the in vitro transcript at the first
generation in the Embodiment 1 was analyzed. As the result, 43% of
these sequences were identical each other among the clone as
separated and extracted. In addition, the gene sequence of the in
vitro transcript at the third generation was analyzed. As the
result, 66% of these sequences were identical each other among
thereof.
[0105] On the other hand, the sequence identity was examined with
regard to the in vitro transcript as obtained in [SELEX method],
instead of [Gel method] in the Embodiment 1. As the result, the
target sequences at the first generation and the third generation
were not able to be detected. Both values were significantly lower
than the values as obtained in the Embodiment 1.
[0106] In addition, the sequence identity of the in vitro
transcript corresponding to the double strand DNA product as
obtained in the Embodiment 2 was examined. As the result, it was
about 66%.
Embodiment 2-1
[0107] The following [Gel method 2] was performed for first
generation, using 50 pM of myeloperoxidase (MPO; sequence number 9)
as the target substance.
[0108] The following [PAGE method] was performed for three
generations (second generation, third generation and fourth
generation in addition to the previous generation), using the
radiorabeled in vitro transcript at the first generation. The
autoradiogram of the obtained polyacrylamide gel at the second
generation, the third generation and the fourth generation are
shown in FIGS. 4, 5 and 6, respectively.
[0109] In FIG. 4, lane P corresponds to the RNA pool only, lanes 1
and 2 correspond to the mouse IgG and the RNA aptamer corresponding
thereto, lane 3 corresponds to yellowish-green fluorescent protein
(CpYGFP; sequence number 15) only, lane 4 corresponds to MPO only,
and lane 5 corresponds to the radiorabeled in vitro transcript as
obtained in use of the mixture of MPO, CpYGFP and MBP,
respectively. In FIG. 5, lane P corresponds to the RNA pool 2 only,
lanes 1 corresponds to the mouse IgG and the RNA aptamer
corresponding thereto, lane 2 corresponds to CpYGFP only, lane 3
corresponds to MPO only, and lane 4 corresponds to the radiorabeled
in vitro transcript as obtained in use of the mixture of MPO,
CpYGFP and MBP, respectively. In FIG. 6, lane I indicates the RNA
aptamer to the mouse IgG, lane II indicates the mouse IgG and the
RNA aptamer corresponding thereto, lane III indicates CpYGFP only,
lane IV indicates the radiorabeled in vitro transcript as obtained
in CpYGFP, and MPO, and lane V indicates the radiorabeled in vitro
transcript as obtained in MPO, and MPO. In addition, in FIGS. 4 to
6, the encircled region with the dashed line indicates the gel as
collected.
[0110] [Preparation 2 of RNA Pool]
[0111] The Preparation 2 of RNA pool was performed as the same as
the above-mentioned [Preparation of RNA pool], except that the DNA
as shown in sequence number 10 was used instead of the DNA as shown
in sequence number 3, to obtain a double strand DNA. Then, a
transcriptional reaction was performed, using T7 RNA polymerase
with so obtained double strand DNA as the template to obtain a RNA
pool 2 (sequence number 11).
[0112] [Gel Method 2]
[0113] 500 pM of the RNA pool 2 was added in a binding buffer 2 (50
mM HEPES/KOH (pH 7.6), 100 mM of NaCl, 5 mM of MgCl.sub.2 and 20%
of glycerol), and the target substance was further added, and stood
for 30 minutes at room temperature.
[0114] The mixture was subjected to the above-mentioned [Gel
method] to obtain an oligonucleotide.
[0115] The steps as mentioned above and the following steps from
the following [RT 2], and the following [PCR 2] and the following
[in vitro transcription] are correctively referred to as a first
generation. In addition, each step of the next generation was
performed, using the in vitro transcript as obtained in the just
before generation, instead of the RNA pool 2.
[0116] [PAGE Method]
[0117] In the above-mentioned [Gel method 2], 500 pM of the RNA
pool was replaced with the radiolabeled in vitro transcript at the
just before generation, 1% agarose gel was replaced with
polyacrylamide (8%; 45 mM of Tris, 0.45 mM of boric acid and 2.5 mM
of MgCl.sub.2), and TBE buffer solution was replaced with a Running
Buffer (45 mM Tris, 0.45 mM of boric acid, and 2.5 mM of
MgCl.sub.2) to perform the electrophoresis for 2 hours at 4.degree.
C. with 160 V (CV).
[0118] While the gel was observed under the exposure with X-ray
film so as to detect the radiorabeled form of the gel after the
electrophoresis, a band in which the gel shift was observed was
cut, and immersed in the buffer solution, RNA molecule was
extracted by means of the free diffusion into the buffer solution,
and was subjected to an appropriate method such as alcohol
precipitation to efficiently obtain an oligonucleotide. In
addition, the oligonucleotide was subjected to the [Gel method
2].
[0119] [RT 2]
[0120] Reverse transcription reaction was performed as the same as
the above-mentioned [RT], except that primer 11 (sequence number
12) was used instead of primer 1 (sequence number 6)
[0121] [PCR 2]
[0122] The PCR 2 was performed as the same as the above-mentioned
[PCR], except that primer 2 (sequence number 7) and primer 3
(sequence number 8) were replaced with primer 12 (sequence number
13) and primer 13 (sequence number 14), respectively to obtain a
double strand DNA product.
Embodiment 2-2
[0123] The in vitro transcript at the fourth generation as obtained
in the Embodiment 2-1 was subjected to the TA cloning, and base
sequences of the cloned 50 colonies were analyzed. As the result,
the oligonucleotides of sequence numbers 16 and 17 were
obtained.
[0124] The radiolabeled in vitro transcripts corresponding to these
oligonucleotides were subjected to the above-mentioned [Binding
Experiment]. Kd value and Bmax of each oligonucleotide were
estimated based on the binding experiment. The result is shown in
Table 3.
TABLE-US-00003 TABLE 3 Kd (nM) Bmax (%) Sequence Number 16 195 52
Sequence Number 17 137.7 77.2
Embodiment 2-3
[0125] The above-mentioned [Binding Experiment] was performed,
using the oligonucleotide of sequence number 16 and the matter
corresponding to the RNA pool 2 as the radiolabeled in vitro
transcript, and using 0, 50 and 100 nM of MPO and 100 nM of bovine
serum albumin (BSA) as the target protein. The result is shown in
Table 4.
TABLE-US-00004 TABLE 4 RNA pool 2 Sequence Number 16 0 nM MPO 1.49
0.24 50 nM MPO 2.66 12.48* 100 nM MPO 1.70 11.44* BSA 1.86 2.46 In
Table 4, each value was obtained in the experiment at n = 3, and
mark * indicates that there is 5% or lower of significant
difference with regard to the value as obtained in the RNA pool
2.
[0126] These results indicate that oligonucleotides capable of
binding to the target substance can be efficiently obtained in the
method for obtaining oligonucleotide according to the present
invention.
[0127] The present invention has been explained in reference to the
preferred embodiment of the present invention. Although the
particular embodiments are illustrated to explain the present
invention, it is apparent that modifications and alterations can be
applied to these particular embodiments without departing from the
spirit and scope of the present invention as defined in the Claims.
That is, it should not be interpreted that the present invention is
limited to the detail of the particular embodiment and the
accompanying drawing.
Sequence CWU 1
1
17174DNAartificialG9-II (10/23) 1gggagaattc cgaccagaag tgctcttaga
atgggactaa gacacgggac aatttcctct 60ctccttcctc ttct
742179PRTEscherichia coli 2Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg
Gly Leu Leu Gly Cys Ile1 5 10 15Ile Thr Ser Leu Thr Gly Arg Asp Lys
Asn Gln Val Glu Gly Glu Val 20 25 30Gln Val Val Ser Thr Ala Thr Gln
Ser Phe Leu Ala Thr Cys Ile Asn 35 40 45Gly Val Cys Trp Thr Val Tyr
His Gly Ala Gly Ser Lys Thr Leu Ala 50 55 60Gly Pro Lys Gly Pro Ile
Thr Gln Met Tyr Thr Asn Val Asp Gln Asp65 70 75 80Leu Val Gly Trp
Pro Ala Pro Pro Gly Ala Arg Ser Met Thr Pro Cys 85 90 95Thr Cys Gly
Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val 100 105 110Ile
Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro 115 120
125Arg Pro Ile Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys
130 135 140Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys
Thr Arg145 150 155 160Gly Val Ala Lys Ala Val Asp Phe Val Pro Val
Glu Ser Met Glu Thr 165 170 175Thr Met Arg397DNAartificialPrimary
pool for preparation of RNA pool 3ccctccacct tgacttcctc tnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn nnnnnntgaa gcgttagcga
gatgcgt 97434DNAartificialPromotor for T7RNA polymerase 4tgtaatacga
ctcactatag gtagatacga tgga 34597RNAartificialRNA pool 5gggaggugga
acugaaggag annnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnacuu cgcaaucgcu cuacgca 97621DNAartificialprimer for RT-PCR
6tgcgtagagc gattgcgaag t 21755DNAartificialprimer for PCR (5')
7tgtaatacga ctcactatag gtagatacga tggagggagg tggaactgaa ggaga
55821DNAartificialprimer for PCR (3') 8acttcgcaat cgctctacgc a
219745PRTHomo sapiens 9Met Gly Val Pro Phe Phe Ser Ser Leu Arg Cys
Met Val Asp Leu Gly1 5 10 15Pro Cys Trp Ala Gly Gly Leu Thr Ala Glu
Met Lys Leu Leu Leu Ala 20 25 30Leu Ala Gly Leu Leu Ala Ile Leu Ala
Thr Pro Gln Pro Ser Glu Gly 35 40 45Ala Ala Pro Ala Val Leu Gly Glu
Val Asp Thr Ser Leu Val Leu Ser 50 55 60Ser Met Glu Glu Ala Lys Gln
Leu Val Asp Lys Ala Tyr Lys Glu Arg65 70 75 80Arg Glu Ser Ile Lys
Gln Arg Leu Arg Ser Gly Ser Ala Ser Pro Met 85 90 95Glu Leu Leu Ser
Tyr Phe Lys Gln Pro Val Ala Ala Thr Arg Thr Ala 100 105 110Val Arg
Ala Ala Asp Tyr Leu His Val Ala Leu Asp Leu Leu Glu Arg 115 120
125Lys Leu Arg Ser Leu Trp Arg Arg Pro Phe Asn Val Thr Asp Val Leu
130 135 140Thr Pro Ala Gln Leu Asn Val Leu Ser Lys Ser Ser Gly Cys
Ala Tyr145 150 155 160Gln Asp Val Gly Val Thr Cys Pro Glu Gln Asp
Lys Tyr Arg Thr Ile 165 170 175Thr Gly Met Cys Asn Asn Arg Arg Ser
Pro Thr Leu Gly Ala Ser Asn 180 185 190Arg Ala Phe Val Arg Trp Leu
Pro Ala Glu Tyr Glu Asp Gly Phe Ser 195 200 205Leu Pro Tyr Gly Trp
Thr Pro Gly Val Lys Arg Asn Gly Phe Pro Val 210 215 220Ala Leu Ala
Arg Ala Val Ser Asn Glu Ile Val Arg Phe Pro Thr Asp225 230 235
240Gln Leu Thr Pro Asp Gln Glu Arg Ser Leu Met Phe Met Gln Trp Gly
245 250 255Gly Leu Leu Asp His Asp Leu Asp Phe Thr Pro Glu Pro Ala
Ala Arg 260 265 270Ala Ser Phe Val Thr Gly Val Asn Cys Glu Thr Ser
Cys Val Gln Gln 275 280 285Pro Pro Cys Phe Pro Leu Lys Ile Pro Pro
Asn Asp Pro Arg Ile Leu 290 295 300Asn Gln Ala Asp Cys Ile Pro Phe
Phe Arg Ser Cys Pro Ala Cys Pro305 310 315 320Gly Ser Asn Ile Thr
Ile Arg Asn Gln Ile Asn Ala Leu Thr Ser Phe 325 330 335Val Asp Ala
Ser Met Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn 340 345 350Leu
Arg Asn Met Ser Asn Gln Leu Gly Leu Leu Ala Val Asn Gln Arg 355 360
365Phe Gln Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn Leu His Asp
370 375 380Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg Ile Pro Cys
Phe Leu385 390 395 400Ala Gly Asp Thr Arg Ser Ser Glu Met Pro Glu
Leu Thr Ser Met His 405 410 415Thr Leu Leu Leu Arg Glu His Asn Arg
Leu Ala Thr Glu Leu Lys Ser 420 425 430Leu Asn Pro Arg Trp Asp Gly
Glu Arg Leu Tyr Gln Glu Ala Arg Lys 435 440 445Ile Val Gly Ala Met
Val Gln Ile Ile Thr Tyr Arg Asp Tyr Leu Pro 450 455 460Leu Val Val
Gly Pro Thr Ala Met Arg Lys Tyr Leu Pro Thr Tyr Arg465 470 475
480Ser Tyr Asn Asp Ser Val Asp Pro Arg Ile Ala Asn Val Phe Thr Asn
485 490 495Ala Phe Arg Tyr Gly His Thr Leu Ile Gln Pro Phe Met Phe
Arg Leu 500 505 510Asp Asn Arg Tyr Gln Pro Met Glu Pro Asn Pro Arg
Val Pro Leu Ser 515 520 525Arg Val Phe Phe Ala Ser Trp Arg Val Val
Leu Glu Gly Gly Ile Asp 530 535 540Pro Ile Leu Arg Gly Leu Met Ala
Thr Pro Ala Lys Leu Asn Arg Gln545 550 555 560Asn Gln Ile Ala Val
Asp Glu Ile Arg Glu Arg Leu Phe Glu Gln Val 565 570 575Met Arg Ile
Gly Leu Asp Leu Pro Ala Leu Asn Met Gln Arg Ser Arg 580 585 590Asp
His Gly Leu Pro Gly Tyr Asn Ala Trp Arg Arg Phe Cys Gly Leu 595 600
605Pro Gln Pro Glu Thr Val Gly Gln Leu Gly Thr Val Leu Arg Asn Leu
610 615 620Lys Leu Ala Arg Lys Leu Met Glu Gln Tyr Gly Thr Pro Asn
Asn Ile625 630 635 640Asp Ile Trp Met Gly Gly Val Ser Glu Pro Leu
Lys Arg Lys Gly Arg 645 650 655Val Gly Pro Leu Leu Ala Cys Ile Ile
Gly Thr Gln Phe Arg Lys Leu 660 665 670Arg Asp Gly Asp Arg Phe Trp
Trp Glu Asn Glu Gly Val Phe Ser Met 675 680 685Gln Gln Arg Gln Ala
Leu Ala Gln Ile Ser Leu Pro Arg Ile Ile Cys 690 695 700Asp Asn Thr
Gly Ile Thr Thr Val Ser Lys Asn Asn Ile Phe Met Ser705 710 715
720Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu Pro Ala Leu
725 730 735Asn Leu Ala Ser Trp Arg Glu Ala Ser 740
7451079DNAartificialDNA sequence used to obtain a RNA pool 2
10agtaatacga ctcactatag gtagatacga tggannnnnn nnnnnnnnnn nnnnnnnnnn
60nnnncatgac gcgcagcca 791179RNAartificialRNA pool 2 11aguaauacga
cucacuauag guagauacga uggannnnnn nnnnnnnnnn nnnnnnnnnn 60nnnncaugac
gcgcagcca 791215DNAartificialprimer 12tggctgcgcg tcatg
151334DNAartificialprimer 13agtaatacga ctcactatag gtagatacga tgga
341415DNAartificialprimer 14catgacgcgc agcca 1515219PRTEscherichia
coli 15Met Thr Thr Phe Lys Ile Glu Ser Arg Ile His Gly Asn Leu Asn
Gly1 5 10 15Glu Lys Phe Glu Leu Val Gly Gly Gly Val Gly Glu Glu Gly
Arg Leu 20 25 30Glu Ile Glu Met Lys Thr Lys Asp Lys Pro Leu Ala Phe
Ser Pro Phe 35 40 45Leu Leu Ser His Cys Met Gly Tyr Gly Phe Tyr His
Phe Ala Ser Phe 50 55 60Pro Lys Gly Thr Lys Asn Ile Tyr Leu His Ala
Ala Thr Asn Gly Gly65 70 75 80Tyr Thr Asn Thr Arg Lys Glu Ile Tyr
Glu Asp Gly Gly Ile Leu Glu 85 90 95Val Asn Phe Arg Tyr Thr Tyr Glu
Phe Asn Lys Ile Ile Gly Asp Val 100 105 110Glu Cys Ile Gly His Gly
Phe Pro Ser Gln Ser Pro Ile Phe Lys Asp 115 120 125Thr Ile Val Lys
Ser Cys Pro Thr Val Asp Leu Met Leu Pro Met Ser 130 135 140Gly Asn
Ile Ile Ala Ser Ser Tyr Ala Arg Ala Phe Gln Leu Lys Asp145 150 155
160Gly Ser Phe Tyr Thr Ala Glu Val Lys Asn Asn Ile Asp Phe Lys Asn
165 170 175Pro Ile His Glu Ser Phe Ser Lys Ser Gly Pro Met Phe Thr
His Arg 180 185 190Arg Val Glu Glu Thr His Thr Lys Glu Asn Leu Ala
Met Val Glu Tyr 195 200 205Gln Gln Val Phe Asn Ser Ala Pro Arg Asp
Met 210 2151660RNAEscherichia coli 16gguagauacg auggaacacg
aagauuucaa agugauaccc cagggcauga cgcgcagcca 601760RNAEscherichia
coli 17gguagauacg auggaacacg aauauuucaa agugauaccc cagggcauga
cgcgcagcca 60
* * * * *